We are interested in studying the molecular mechanism involved in retinoblastoma formation. Recently we have found that the N-myc gene is amplified in three independent cases of retinoblastoma and expressed at a high level in most of the primary retinoblastoma examined. One of our main goals is to elucidate how the retinoblastoma susceptibility gene (Rb gene) interacts with cellular proto-oncogenes to cause a tumor. We plan to elucidate the structure and function of the N-myc gene and to clone the Esterase-D gene for the purpose of locating the putative Rb gene. We shall first attempt to clone the complete N-myc genes either from mRNA or from genomic DNA of different retinoblastomas. Transforming potential of such clones will be explored by transfecting them into human embryo retina cells (HER) or NIH/3T3 cells. We expect that some of the N-myc genes will carry a regulatory alteration since we have observed a high level of N-myc mRNA expressed in some retinoblastomas in which the N-myc gene was not amplified. We plan to investigate such regulatory changes in detail. Second, we will examine the expression of the N-myc gene in the developmental process of fetal retina by in situ hybridization with a complementary DNA probe and by immunocytochemistry with N-myc antisera. We would like to determine whether the induction of differentiation in retinoblastoma cells results in a decrease of N-myc expression. Third, we want to test whether oncogenic viruses, such as RNA tumor viruses, can induce retinoblastoma in vitro and to compare the role of the N-myc gene in virally induced and naturally occurring retinoblastomas. It will be of interest to examine the behavior of the N-myc gene in such virally induced retinoblastomas. The overall results from the first three parts should clarify the role that the N-myc gene plays in retinoblastoma formation. Fourth, we shall generate antisera against either the synthetic N-myc peptides or the N-myc protein expressed in E. coli. We shall use these antisera to characterize the N-myc gene product in detail. In the final part, we shall clone the Esterase-D gene using oligonucleotides synthesized according to the nucleotide sequences deduced from amino acid sequences as a probe. After the clone is identified, we shall use it to locate physically the putative retinoblastoma gene(s) by chromosomal walking.